Supplementation of all trans retinoic acid ameliorates ethanol-induced endoplasmic reticulum stress.

CONTEXT: Molecular pathogenesis of chronic alcoholism is linked to increased endoplasmic reticulum stress. Ethanol is a competitive inhibitor of vitamin A metabolism and vitamin A supplementation aggravates existing liver problems. Hence, we probed into the impact of supplementation of all trans retinoic acid (ATRA), the active metabolite of vitamin A on ethanol-induced endoplasmic reticulcum stress. METHODS: Male Sprague-Dawley rats were divided into four groups - I: Control; II: Ethanol; III: ATRA; IV: ATRA + Ethanol. After 90 days the animals were sacrificed to study markers of lipid peroxidation in hepatic microsomal fraction and expression of ER stress proteins and apoptosis in liver. RESULTS AND CONCLUSION: Ethanol caused hepatic hyperlipidemia, enhanced microsomal lipid peroxidation, upregulated expression of unfolded protein response associated proteins and that of apoptosis. Ethanol also led to downregulation of retinoid receptors. ATRA supplementation reversed all these alterations indicating the decrease in ethanol-induced endoplasmic reticulum stress.

The preventive effects of taurine on neural tube defects through the Wnt/PCP-Jnk-dependent pathway.

The aim of this study was to clarify the protective role of taurine in neuronal apoptosis and the role of the Wnt/PCP-Jnk pathway in mediating the preventive effects of taurine on neural tube defects (NTDs). HT-22 cells (a hippocampal neuron cell line) were divided into a control group, a glutamate-induced apoptosis group, and glutamate (4.0 mmol/L) plus low-dose taurine (L; 0.5 mmol/L) and high-dose taurine (H; 2.0 mmol/L) groups. The MTT assay was used to monitor cell proliferation and cell survival. Immunofluorescence and Western blot analyses were used to determine caspase 9 expression. Retinoic acid (RA) induced embryonic NTDs in Kunming mice, thus establishing an NTD model. Pregnant mice were divided into a control group, an RA (30 mg/kg body weight) group, and an RA (30 mg/kg body weight) plus taurine (free drinking of 2 g/L solution) group. Immunohistochemistry and Western blot analyses were used to detect the expression of Dvl, RhoA and phosphorylated (p)-Jnk/Jnk in the embryonic neural tubes. In HT-22 cells, the apoptosis rate was significantly higher and caspase 9 activation was also significantly increased in the glutamate-induced apoptosis group compared to the L and H taurine groups. In the NTD model, the expression levels of Dvl, RhoA, and p-Jnk were significantly higher in the RA group than in the control group, whereas they were significantly reduced in the RA + taurine group. This study suggests that taurine has positive effects on neuronal protection and NTD prevention. Moreover, the Wnt/PCP-Jnk-dependent pathway plays an important role in taurine-mediated prevention of NTDs.

Nutrition and psoriasis.

Nutritional supplementation may provide a viable treatment alternative in patients with psoriasis. Randomized, controlled trials have shown the effectiveness of topical vitamin A and D derivatives, intravenous ω-3 fatty acids, oral inositol, and various combined therapies. Dual therapies of ultraviolet B phototherapy and fish oil, retinoids and thiazolidinediones, and cyclosporine and a low-calorie diet were effective in the treatment of psoriasis in randomized, controlled trials. This contribution also reviews the potential negative effect of alcohol and the potential positive effects of vitamin B(12), selenium, retinoic acid metabolism-blocking agents, and a gluten-free diet in the treatment of psoriasis.

Hif1α down-regulation is associated with transposition of great arteries in mice treated with a retinoic acid antagonist.

BACKGROUND: Congenital heart defect (CHD) account for 25% of all human congenital abnormalities. However, very few CHD-causing genes have been identified so far. A promising approach for the identification of essential cardiac regulators whose mutations may be linked to human CHD, is the molecular and genetic analysis of heart development. With the use of a triple retinoic acid competitive antagonist (BMS189453) we previously developed a mouse model of congenital heart defects (81%), thymic abnormalities (98%) and neural tube defects (20%). D-TGA (D-transposition of great arteries) was the most prevalent cardiac defect observed (61%). Recently we were able to partially rescue this abnormal phenotype (CHD were reduced to 64.8%, p = 0.05), by oral administration of folic acid (FA). Now we have performed a microarray analysis in our mouse models to discover genes/transcripts potentially implicated in the pathogenesis of this CHD. RESULTS: We analysed mouse embryos (8.5 dpc) treated with BMS189453 alone and with BMS189453 plus folic acid (FA) by microarray and qRT-PCR. By selecting a fold change (FC) ≥ ± 1.5, we detected 447 genes that were differentially expressed in BMS-treated embryos vs. untreated control embryos, while 239 genes were differentially expressed in BMS-treated embryos whose mothers had also received FA supplementation vs. BMS-treated embryos. On the basis of microarray and qRT-PCR results, we further analysed the Hif1α gene. In fact Hif1α is down-regulated in BMS-treated embryos vs. untreated controls (FCmicro = -1.79; FCqRT-PCR = -1.76; p = 0.005) and its expression level is increased in BMS+FA-treated embryos compared to BMS-treated embryos (FCmicro = +1.17; FCqRT-PCR = +1.28: p = 0.005). Immunofluorescence experiments confirmed the under-expression of Hif1α protein in BMS-treated embryos compared to untreated and BMS+FA-treated embryos and, moreover, we demonstrated that at 8.5 dpc, Hif1α is mainly expressed in the embryo heart region. CONCLUSIONS: We propose that Hif1α down-regulation in response to blocking retinoic acid binding may contribute to the development of cardiac defects in mouse newborns. In line with our hypothesis, when Hif1α expression level is restored (by supplementation of folic acid), a decrement of CHD is found. To the best of our knowledge, this is the first report that links retinoic acid metabolism to Hif1α regulation and the development of D-TGA.

Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol.

BACKGROUND: A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reduction defects as the teratogenic end point. METHODS: Ethanol, Disulfiram, or BMS-189453 was administered to C57BL/6J mice on the 9(th) day of pregnancy. Forelimb morphology was assessed on gestation day 18 using Alcian blue and Alizarin red staining. Nile blue sulfate or LysoTracker Red (LTR) vital staining identified cell death in the limb bud. The ability of RA to prevent ethanol-induced cell death was assessed by coadministration followed by laser scanning confocal microscopic examination of LTR-staining. In situ hybridization and qPCR were used to examine gene expression in treated limb buds. RESULTS: Ethanol, Disulfiram, and BMS-189453 resulted in postaxial ectrodactyly, intermediate ectrodactyly, and other digital defects. Excessive Nile blue sulfate staining was evident in the presumptive AER following each of the three exposures. Ethanol-induced LTR staining was prevented by RA supplementation. Both in situ hybridization and qPCR illustrated decreases in Shh and Tbx5 in ethanol-exposed embryos as compared to control. CONCLUSIONS: Contrary to studies of prolonged RA deficiency, acute exposure to functional antagonists of RA results in limb defects that are morphologically similar to those caused by ethanol. The rescue of ethanol-induced cell death by RA and similar changes in Shh transcription further suggest that RA contributes to ethanol-induced limb dysmorphology. Moreover, the repression of key mediators of limb development soon after ethanol exposure adds to the existing knowledge of the pathogenic effects of ethanol.

Retinoic acid stimulates the cell cycle machinery in normal T cells: involvement of retinoic acid receptor-mediated IL-2 secretion.

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27(Kip1), increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.

Differential expression of chicken CYP26 in anterior versus posterior limb bud in response to retinoic acid.

Multiple studies indicate that quantitative control of the levels of all-trans-retinoic acid (RA) in the vertebrate embryo is necessary for correct development. The function of RA in cells is regulated by a number of coordinated mechanisms. One of those mechanisms involves controls on the rate of RA catabolism. Recently, enzymes capable of catabolizing RA were found to constitute a new family, called CYP26, within the cytochrome P450 superfamily. CYP26 homologues have been isolated from human, mouse, zebra fish, and recently from the chick. In this study, we examined the regulation of chicken CYP26 (cCYP26) expression by RA during the early phase of chick limb outgrowth. In the anterior limb mesenchyme and apical ectodermal ridge (AER), cCYP26 expression was induced in a concentration dependent manner by implanting beads soaked in 0.1, 1, and 5 mg/ml RA. The RA-induced expression of cCYP26 in anterior limb mesenchyme and the AER was detected as early as 1 hr after treatment and was not affected by the presence of cycloheximide. In contrast to the anterior limb, the induction of cCYP26 was dramatically reduced (or absent) when RA beads were implanted in the posterior limb mesenchyme. Furthermore, induction of cCYP26 expression in the anterior mesenchyme was inhibited by transplantations of the zone of polarizing activity (ZPA) and by Shh-soaked beads. Our data suggest that different mechanisms regulate retinoid homeostasis in the AER and mesenchyme during limb bud outgrowth. J. Exp. Zool. 290:136-147, 2001.

Differential effects of retinoic acid and a retinoid antagonist on the spatial distribution of the homeoprotein Hoxb-7 in vertebrate embryos.

An antibody raised against the recombinant Xenopus laevis Hoxb-7 protein (López and Carrasco [1992] Mech. Dev. 36:153-164) recognizes the 30 kDa translation product of the Hoxb-7 gene in X. laevis and the cognate nuclear protein in chicken embryos. The X. laevis Hoxb-7 protein was expressed maternally and zygotically. Treatment of X. laevis and chicken embryos with either all-trans retinoic acid (RA) or the retinoid antagonist Ro 41-5253 (Ro; Apfel et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:7129-7133) during early development induced malformations of the neural tube and complementary changes in the expression domain of the homeoprotein Hoxb-7. Treatment of X. laevis embryos with retinoic acid during gastrulation induced an anterior shift of the Hoxb-7 expression domain and was correlated with an enlargement of rhombomere r7. In addition to a reduction in rhombomere numbers and of forebrain size, various malformations involving all three germ layers were observed. Treatment of X. laevis embryos with the antagonist Ro before or during gastrulation caused a progressive reduction of the Hoxb-7 domain and also dose-dependent malformations of all three germ layers. RA or Ro treatment of chicken embryos from the beginning of gastrulation caused changes of the Hoxb-7 expression domain very similar to those observed in X. laevis. In particular, either a dose-dependent loss of the Hoxb-7 protein in the neural tube or an ectopic expression in the forebrain region was observed. The results of this study indicate that endogenous retinoids regulate the spatial expression of homeobox-containing genes in vertebrates.

Regulation of estradiol 17 beta-hydroxysteroid dehydrogenase expression and activity by retinoic acid in T47D breast cancer cells.

Estradiol 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) mediates the interconversion of estrone and estradiol in endocrine-responsive tissues such as the breast. The control of 17 beta HSD expression by all-trans-retinoic acid (RA) in T47D breast cancer cells was examined using a specific 17 beta HSD complementary DNA probe. Two main 17 beta HSD messenger RNA (mRNA) transcripts of 2.2 and 1.3 kilobases (kb) were detected, of which only the 1.3-kb mRNA was regulated. RA increased expression of the 17 beta HSD 1.3-kb mRNA in a dose- and time-dependent manner, and the increased expression of this mRNA by RA was inhibited by a 10-fold excess of a RA antagonist Ro 41-5253. Insulin-like-growth factor-I, interleukin-1, and estradiol, previously shown to increase 17 beta HSD activity in breast cancer cells, had little effect on 17 beta HSD gene expression. To relate the effect of increased 17 beta HSD 1.3-kb mRNA expression to 17 beta HSD activity, the conversion of estrone to estradiol (reductive) and that of estradiol to estrone (oxidative) were measured in intact T47D cell monolayers. Whereas RA increased 17 beta HSD reductive activity, it had no effect on oxidative activity. The addition of excess NAD increased 17 beta HSD oxidative activity in control and RA-treated cells, but the addition of NADH had no effect on 17 beta HSD reductive activity. These results suggest that the increased expression of the 17 beta HSD 1.3-kb mRNA induced by RA is associated with an increase in 17 beta HSD reductive activity, but that endogenous cofactor levels may determine the direction in which this enzyme acts in T47D cells.

Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoic acid-induced granulocytic differentiation.

HL60 cells are devoid of endogenous epidermal growth factor receptor (EGFR). They respond to retinoic acid and undergo terminal granulocytic differentiation. EGFR complementary DNA was introduced into HL60 cells by retroviral gene transfer. Scatchard plot showed that the binding characteristics are identical to those of A431 cells. HL60-EGFR cells were estimated to express 34,000 EGFR/cell (Kd = 5 nM). The tyrosine phosphorylation upon ligand binding is the first step of signal transduction. The dominant phosphotyrosyl proteins in epidermal growth factor-stimulated HL60-EGFR cells include a 170 kDa protein (EGFR itself), and 125 and 53 kDa proteins. The EGFR signal results in the induction of 92 kDa gelatinase/matrix metalloproteinase in HL60-EGFR cells, thereby providing evidence of the function of the exogenous EGFR and a semiquantitative measure of the EGFR signal. These HL60-EGFR cells offer a unique opportunity to examine the potentially important role of EGFR (c-erbB) in maintaining homeostasis between self-renewal and differentiation. c-erbB has been shown to play a physiological role in the self-renewal of the very early avian stem cells which do express EGFR. The v-erbB (double truncated EGFR) has been shown to cause avian erythroblastosis. We found that these HL60-EGFR cells responded to retinoic acid differently from the HL60-control cells. A partial block of only 45% granulocytic differentiation and concomitant proliferation was noted, consistent with a shift of balance between self-renewal and differentiation toward the former.

Suppression by cyclohexanetriones of retinoic acid-induced cartilage degradation in vitro and teratogenicity in vivo.

In cultured fetal rat bones, cyclohexanetriones that stimulate prostaglandin synthesis inhibited retinoic acid-induced cartilage degradation in a dose-dependent manner. The inhibition by the cyclohexanetrione Ro 31-0521 was reversible, indicating that the effect was not due to cytotoxicity. Excess retinoic acid is teratogenic in rats and adversely affects the normal differentiation of various morphogenetic systems, depending on the time of administration. The following retinoic acid-induced malformations were suppressed by Ro 31-0521: malformations of long bones and of apical phalanges induced on days 13 and 15 of gestation, respectively; spina bifida and tail malformations induced on day 11 of gestation and cleft palate induced on day 15 of gestation. However, cleft palate and other head malformations including exencephaly induced by retinoic acid on day 11 of gestation were not suppressed but even increased by Ro 31-0521. At a high dose, Ro 31-0521 given alone on day 11 of gestation was embryolethal and teratogenic but was not on the tested other days, indicating that the cyclohexanetrione at specific stages and doses also interfered with normal morphogenesis like retinoic acid. Assuming that stimulation of prostaglandin synthesis is the main biological effect of the cyclohexanetriones, our findings suggest that prostaglandins may be involved in mediating retinoid action.